Human monocytic ehrlichiosis (HME) caused by Ehrlichia chaffeensis and human granulocytic ehrlichiosis (HGE) caused by Anaplasma phagocytophila, are emerging infectious diseases. These bacteria are fastidious obligatory intracellular bacteria that require repeated transmission between two vastly different hosts: vertebrates and invertebrates. How these bacteria enter and continue to thrive within hostile host milieu such as neutrophils or the tick vector is largely unknown. We recently characterized genes encoding a type IV secretion system (T4SS) and two component-regulatory system (2CRS) in HGE and HME agents. This proposal aims to understand the roles of T4SS and 2CRS in pathogenesis of HME and HGE. The specific aims of this project are as follows: 1. Examine the colocalization of T4SS proteins expressed by individual E. chaffeensis and A. phagocytophila organisms with endosome-lysosome markers and signaling molecules in human leukocytes during attachment, internalization, and subsequent proliferation, and in response to environmental stimuli. 2. Characterize expression of T4SS in ticks and the skin of mammals during tick transmission. 3. Identify T4SS effector or substrate molecules. 4. Characterize the 2CRS of E. chaffeensis and A. phagocytophila by expressing recombinant proteins and studying the phosphorylation of sensor kinases and response regulators in vitro, and in vivo under controlled sets of environmental conditions; by examining the effects of 2CRS (histidine kinase) inhibitors on phenotypes of E. chaffeensis and A. phagocytophila; and by studying genes regulated by 2CRS. 5. Construct novel mutants ofA. phagocytophila and E. chaffeensis in which a predicted critical gene is disrupted, or conditionally silenced to determine the functions of T4SS and 2CRS. The data obtained from this study will provide a new perspective for understandings the dynamic signaling events between obligatory intracellular bacteria and their mammalian and tick hosts. This project will potentially provide a technical breakthrough in this field of research. The results may point to a potential target for new treatment and prevention of HME and HGE. [unreadable] [unreadable]